Thes4A strand insertion in intact, cleaved, and complexed alpha1-antichymotrypsin

Quihui Zhong, University of Pennsylvania

Abstract

This thesis is directed toward understanding the mechanism of the interaction between $\alpha$1-antichymotrypsin (ACT), a member of serine protease inhibitors (serpins) superfamily, and target proteases. The focus is on how the ACT activity is regulated by the s4A strand insertion into $\beta$-sheet A. The relationship between inhibitory activities of intact forms and stabilities of cleaved forms of ACT is investigated by constructing ACT variants: T345R, A347R, A350R, A352R, and I355R. The interaction of variants and chymotrypsin (Chtr) is characterized by the rate constant of inhibition (ki), stoichiometry of inhibition (SI), and the lifetime of the Chtr/ACT complex. A350R and I355R inhibit Chtr with similar ki and SI (10$\sp6$M$\rm\sp{-1}s\sp{-1}$, SI = 1.0) as wt-ACT whereas A352R inhibits Chtr with similar ki, but a higher SI about 5. T345R and A347R are substrates of Chtr. Unfolding patterns of intact and cleaved forms as a function of GuHCl are measured by circular dichroism. The unfolding profiles of intact forms superimpose, each undergoing a sharp transition centered at about 3 M GuHCl. However, the unfolding profiles of cleaved forms display a spectrum of transitions ranging from 4 to 7.5 M GuHCl. The order of stability for each cleaved variant is: WT $>$ I355R $>$ A352R $>$ A350R $>$ A347R $>$ T345R. The enhanced stability of cleaved form provides evidence that the Are residue at the hinge does not prevent s4A strand insertion after cleavage; however, the Arg residue within the hinge region destroys inhibitory activity by destablizing cleaved serpins. The conformation of protease/serpin complex is probed by comparing the stabilities of intact, cleaved, and complexed ACT in heat and urea. To avoid the artifact of s4A strand insertion induced by earlier denaturation of Chtr, which is completely denatured at 3 M GuHCl, a thermally stable protease, aqualysin I (AqI) is chosen as the ACT partner. Incubating the AqI/ACT complex at high temperatures following centrifugation results in the release of active AqI and stable cleaved ACT The complex shows no transition on TUG gel, similar to what is seen for cleaved ACT. Taken together, these results indicate that the conformation of complexed ACT is more like cleaved ACT in terms of s4A strand insertion.

Subject Area

Biochemistry|Molecular biology|Biophysics

Recommended Citation

Zhong, Quihui, "Thes4A strand insertion in intact, cleaved, and complexed alpha1-antichymotrypsin" (1996). Dissertations available from ProQuest. AAI9628037.
https://repository.upenn.edu/dissertations/AAI9628037

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